To study the possibility of cross-species transmission of Chronic Wasting Disease (CWD), two species of nonhuman primates, squirrel monkeys and cynomolgus macaques, were infected orally or intracerebrally with brain material derived from CWD-affected deer or elk. All of the squirrel monkeys developed clinical neurological signs and were confirmed by biochemical and pathological testing of brain to have a prion disease. In contrast, experiments done in cynomolgus macaques, which are genetically closer to humans than are squirrel monkeys, were not susceptible to CWD by either route of inoculation. To test for a subclinical level of infection in the macaques we screened brain, spinal column and lymphoid tissues for evidence prion infection by several methods including IHC for prion protein, H&E for neuropathology, immunoblot (for disease associated prion protein). Although our previous studies showed no convincing evidence of CWD infection of cynomolgus macaques, occasional older CWD-injected animals showed unusual areas of PrP staining in brain. Therefore, in FY18, we screened additional age-matched uninfected cynomolgus macaques using immunohistochemistry for detection of abnormal PrP. In these studies, several abnormal forms of PrP staining appeared to be similar in both CWD-injected and uninjected macaques, thus indicating that these types of PrP staining were related to PrP changes associated with aging and were not evidence for CWD infection. In FY18, we also used the newly developed RT-QuIC test for PrP amyloid seeding which has been correlated with the presence of infectious prions in humans and numerous animal models. None of the uninjected or CWD-injected cynomolgus macaques were positive by RT-QuIC testing. Thus, after 13 years, no evidence for CWD transmission to macaques was detected clinically or by using several sensitive prion disease-screening assays in our studies. Our data showed strong species-specific differences in susceptibility of two species of non-human primates to CWD. Based on the closer genetic relationship between humans and cynomolgus macaques, and previous work demonstrating that macaques have a similar prion disease susceptibility pattern to humans, these data suggest that humans may also be resistant to CWD infection. In FY19 we tested CWD transmission to two lines of transgenic mice that expressed human prion protein. The mice expressed very high levels of prion protein and are susceptible to many human prion diseases. Following very long observation periods, no mice developed signs of disease consistent with prion infection. Brains were screened for sub-clinical transmission by IHC, WB and RT-QuIC. A small number of the brains had weak positive RT-QuIC results. These suspicious mice are being further characterized by performing an additional passage in transgenic mice. We are also studying how long prions can persist in brain following inoculation, in the absence of any additional replication. The ability of infectious prions to remain uncleared from brain may explain our previous suspect results.